In various human pathologies there are countless alterations in platelet production or function. Yet many of these pathogenesis and the required targeted therapies remain unknown, resulting in palliative treatments. In vivo, megakaryocytes associate with the bone marrow microvasculature where they extend proplatelets that protrude through the vascular endothelium into the lumen and release platelets into the blood stream. The scientific and clinical communities are actively searching for new modes to generate functional platelets ex vivo to address clinical needs as well as for insight into fundamental studies of mechanisms. We hypothesize that engineering a 3D bone marrow mimic, as demonstrated in our current grant, will propel mechanistic understanding of platelet shedding and determine future protocols for therapeutic inquiry. To test our hypothesis, in Aim 1 we will utilize non-thrombogenic silk protein biomaterial in a modified ex vivo three dimensional (3D) tissue model of the bone marrow to study platelet release from megakaryocytes derived from human induced pluripotent stem cells (hiPSCs); to compare with the results in the current grant using megakaryocytes from umbilical cord blood progenitors.
In Aim 2 we will focus on the use of the bioreactor systems to study the effects of thrombopoietin mimetics on human megakaryocytes derived from patients affected by inherited thrombocytopenias and healthy controls.
In Aim 3 we will conduct studies to assess the functionality of the platelets released in the bioreactor systems in vitro and in vivo. The outcome of these studies is expected to be unprecedented insight into mechanisms that control platelet formation. These insights will build on our ability to generate functional human platelets ex vivo but with significant improvements in cell sources, disease insight and functional assessments in this renewal proposal. The development of a unified solution based on the proposed science and technologies will clarify the impact of thrombopoietin mimetics on human megakaryocyte behaviour in terms of activation of intracellular signaling, differentiation, interaction with the extracellular environment and platelet production, all with major implications for human health. Importantly, the successful outcome of this project will provide researchers with new specialized tools for predicting the efficacy and safety of new drugs to address megakaryocyte- related diseases. In addition, the results will provide an important next step towards clinically relevant sources and supplies of functional human platelets for patient treatments.
Megakaryocytes in the bone marrow are responsible for the continuous production of platelets in the blood. Under- or over-production of platelets has major clinical implications for many diseases, including thrombocytopenia and myeloproliferative neoplasms, where life-threatening side effects with incurable outcomes are common. The broader impact of this work is the design of new tools to mimic the bone marrow vascular niche ex vivo that enable: (1) insight into mechanisms controlling platelet release in a physiological relevant manner, and (2) testing of candidate drug treatments for platelet-related diseases using patient derived cells.
| Tozzi, Lorenzo; Laurent, Pierre-Alexandre; Di Buduo, Christian A et al. (2018) Multi-channel silk sponge mimicking bone marrow vascular niche for platelet production. Biomaterials 178:122-133 |
| Di Buduo, C A; Kaplan, D L; Balduini, A (2017) In vitro generation of platelets: Where do we stand? Transfus Clin Biol 24:273-276 |
| Abbonante, Vittorio; Di Buduo, Christian Andrea; Gruppi, Cristian et al. (2017) A new path to platelet production through matrix sensing. Haematologica 102:1150-1160 |
| Pouli, Dimitra; Tozzi, Lorenzo; Alonzo, Carlo A et al. (2017) Label free monitoring of megakaryocytic development and proplatelet formation in vitro. Biomed Opt Express 8:4742-4755 |
| Di Buduo, Christian A; Soprano, Paolo M; Tozzi, Lorenzo et al. (2017) Modular flow chamber for engineering bone marrow architecture and function. Biomaterials 146:60-71 |
| Balduini, Alessandra; Di Buduo, Christian A; Kaplan, David L (2016) Translational approaches to functional platelet production ex vivo. Thromb Haemost 115:250-6 |
| Di Buduo, Christian A; Currao, Manuela; Pecci, Alessandro et al. (2016) Revealing eltrombopag's promotion of human megakaryopoiesis through AKT/ERK-dependent pathway activation. Haematologica 101:1479-1488 |
| Abbonante, Vittorio; Di Buduo, Christian A; Gruppi, Cristian et al. (2016) Thrombopoietin/TGF-?1 Loop Regulates Megakaryocyte Extracellular Matrix Component Synthesis. Stem Cells 34:1123-33 |
| Currao, Manuela; Malara, Alessandro; Di Buduo, Christian A et al. (2016) Hyaluronan based hydrogels provide an improved model to study megakaryocyte-matrix interactions. Exp Cell Res 346:1-8 |
| Di Buduo, Christian A; Wray, Lindsay S; Tozzi, Lorenzo et al. (2015) Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies. Blood 125:2254-64 |
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